Identification of SLAMF3 (CD229) as an inhibitor of hepatocellular carcinoma cell proliferation and tumour progression.

Although hepatocellular carcinoma (HCC) is one of the most common malignancies and constitutes the third leading cause of cancer-related deaths, the underlying molecular mechanisms are not fully understood. In the present study, we demonstrate for the first time that hepatocytes express signalling lymphocytic activation molecule family member 3 (SLAMF3/CD229) but not other SLAMF members. We provide evidence to show that SLAMF3 is involved in the control of hepatocyte proliferation and in hepatocellular carcinogenesis. SLAMF3 expression is significantly lower in primary human HCC samples and HCC cell lines than in human healthy primary hepatocytes. In HCC cell lines, the restoration of high levels of SLAMF3 expression inhibited cell proliferation and migration and enhanced apoptosis. Furthermore, SLAMF3 expression was associated with inhibition of HCC xenograft progression in the nude mouse model. The restoration of SLAMF3 expression levels also decreased the phosphorylation of MAPK ERK1/2, JNK and mTOR. In samples from resected HCC patients, SLAMF3 expression levels were significantly lower in tumorous tissues than in peritumoral tissues. Our results identify SLAMF3 as a specific marker of normal hepatocytes and provide evidence for its potential role in the control of proliferation of HCC cells.

Locally expressed IGF1 propeptide improves mouse heart function in induced dilated cardiomyopathy by blocking myocardial fibrosis and SRF-dependent CTGF induction.

Cardiac fibrosis is critically involved in the adverse remodeling accompanying dilated cardiomyopathies (DCMs), which leads to cardiac dysfunction and heart failure (HF). Connective tissue growth factor (CTGF), a profibrotic cytokine, plays a key role in this deleterious process. Some beneficial effects of IGF1 on cardiomyopathy have been described, but its potential role in improving DCM is less well characterized. We investigated the consequences of expressing a cardiac-specific transgene encoding locally acting IGF1 propeptide (muscle-produced IGF1; mIGF1) on disease progression in a mouse model of DCM [cardiac-specific and inducible serum response factor (SRF) gene disruption] that mimics some forms of human DCM. Cardiac-specific mIGF1 expression substantially extended the lifespan of SRF mutant mice, markedly improved cardiac functions, and delayed both DCM and HF. These protective effects were accompanied by an overall improvement in cardiomyocyte architecture and a massive reduction of myocardial fibrosis with a concomitant amelioration of inflammation. At least some of the beneficial effects of mIGF1 transgene expression were due to mIGF1 counteracting the strong increase in CTGF expression within cardiomyocytes caused by SRF deficiency, resulting in the blockade of fibroblast proliferation and related myocardial fibrosis. These findings demonstrate that SRF plays a key role in the modulation of cardiac fibrosis through repression of cardiomyocyte CTGF expression in a paracrine fashion. They also explain how impaired SRF function observed in human HF promotes fibrosis and adverse cardiac remodeling. Locally acting mIGF1 efficiently protects the myocardium from these adverse processes, and might thus represent a therapeutic avenue to counter DCM.

Transgenic expression of human INS gene in Ins1/Ins2 double knockout mice leads to insulin underproduction and diabetes in some male mice.

We have generated transgenic mouse lines expressing exclusively a human INS transgene on an Ins1/Ins2 double knockout (mIKO) background. The transgene expression was driven by either a 4000 bp or a 353 bp promoter. These transgenic lines, designated mIKO:INS4000 and mIKO:INS353, were viable and fertile. Determination of the amounts of insulin transcripts and total pancreatic insulin content revealed relative insulin underproduction in both lines, from birth to adulthood. Total pancreatic insulin stores in mIKO:INS4000 and mIKO:INS353 mice represented only about 50% and 27%, respectively, as compared to wild-type mice. Morphometric analysis of pancreas did not show any compensatory beta-cell hyperplasia. The majority of animals in both lines remained normoglycemic throughout their lives. Nevertheless, glucose tolerance tests revealed glucose intolerance in nearly half of mIKO:INS4000 male mice, likely due to impaired insulin secretion detected in those animals. In addition, a small fraction (2-4%) of male mice in both lines spontaneously developed diabetes with very distinct pathophysiological features. Diabetes was never seen in female animals. The diabetes developed by mIKO:INS353 mice was rapidly lethal, accompanied by a dramatic depletion of pancreatic insulin stores whereas the mIKO:INS4000 diabetic animals could live for several months. This suggests a possible link between the structure of the human INS gene promoter and the type of diabetes developed in these lines.

Conditional inactivation of the murine serum response factor in the pancreas leads to severe pancreatitis.

The Serum Response Factor (SRF) is widely expressed transcription factor acting at the confluence of multiple signaling pathways and has been implicated in the control of differentiation, growth, and cell death. In the present study, we found that SRF is expressed in the developing and adult pancreas. To explore the possible role of SRF in this organ, we have generated mutant mice with conditional disruption of the Srf gene. Such mutants presented normal development of both the exocrine and endocrine pancreas indicating that SRF is dispensable for pancreas ontogenesis. However, after weaning, these mice developed profound morphological alterations of the exocrine pancreas, which were reminiscent of severe pancreatitis. In these mice, massive acinar injury, Nuclear Factor Kappa B activation and proinflammatory cytokines release led to complete destruction of the exocrine pancreas and its replacement by adipose tissue. Despite these changes, the organization and function of the endocrine islets of Langerhans remained well-preserved. This new animal model of spontaneous pancreatitis could prove a valuable tool to gain further insight into the physiopathology of this disease.

Interplay between FGF10 and Notch signalling is required for the self-renewal of pancreatic progenitors.

Recent studies have shown that persistent expression of FGF10 in the developing pancreas of transgenic mice results in enhanced and prolonged proliferation of pancreatic progenitors, pancreatic hyperplasia and impaired pancreatic differentiation. These studies have also suggested that FGF10 prevents the differentiation of pancreatic progenitors by maintaining persistent Notch signalling. Here, we provide experimental evidence sustaining the capacity of FGF10 to induce the proliferation of pancreatic precursors, while preventing their differentiation. Using explant cultures of E10.5 isolated dorsal pancreatic epithelium, we found that FGF10 maintained Notch activation and induced the expansion of pancreatic precursors while blocking their differentiation. In addition, by using a gamma-secretase inhibitor, we were able to down-regulate the expression of Hes1, a target gene of the Notch pathway in explant cultures of pancreatic epithelium treated with FGF10. In such explants, the effect of FGF10 on the proliferation and maintenance of pancreatic progenitors was suppressed. These results demonstrate that activation of the Notch pathway is required as a downstream mediator of FGF10 signalling in pancreatic precursor cells.

Knock-in of diphteria toxin A chain gene at Ins2 locus: effects on islet development and localization of Ins2 expression in the brain.

We report here knock-in of diphteria toxin A chain (dta) gene at the Ins2 locus, using the strategy previously employed to insert lacZ under control of the Ins2 promoter. Mutant Ins2(dta/+), Ins2(dta/lacZ) or Ins2(lacZ/+) mouse pups were generated by breeding and analyzed to study the effects of toxigenetic beta-cell ablation on islet development and to localize the extrapancreatic Ins2 expression site in the brain. Ins2(dta/+) and Ins2(dta/lacZ) pups developed a severe diabetic ketoacidosis and died rapidly. Histological analysis of their pancreas revealed that beta-cells completely disappeared in their islets as evidenced by loss of lacZ activity or insulin immunonostaining. beta-cell ablation did not alter the size of other islet cell populations which were normal at birth, although the glucagon-cell population was reduced by 85% at embryonic day E12.5. In the brain, comparative analysis of lacZ expression in Ins2(lacZ/+) and Ins2(dta/laZ) mice identified the choroid plexus (CP) as a major Ins2 expression site. This finding was confirmed by RT-PCR analysis of insulin transcripts in RNAs prepared from microdissected wild-type CP. Transcripts for other key beta-cell markers, with the notable exception of Pdx-1, were also found in CP RNAs. These results must revive interest in studies focused on extrapancreatic insulin gene expression.